The role of M1 muscarinic receptor agonism of N-desmethylclozapine in the unique clinical effects of clozapine.

RATIONALE: Clozapine is a unique antipsychotic, with efficacy against positive symptoms in treatment-resistant schizophrenic patients, and the ability to improve cognition and treat the negative symptoms characteristic of this disease. Despite its unique clinical actions, no specific molecular mechanism responsible for these actions has yet been described. OBJECTIVES AND METHODS: To comprehensively profile a large library of neuropsychiatric drugs, including most antipsychotics, at human monoamine receptors using R-SAT, an in vitro functional assay. RESULTS: Profiling revealed that N-desmethylclozapine (NDMC), the principal metabolite of clozapine, but not clozapine itself, is a potent and efficacious muscarinic receptor agonist, a molecular property not shared by any other antipsychotic. To further explore the role of NDMC muscarinic receptor agonist properties in mediating the physiological actions of clozapine, systemically administered NDMC was found to stimulate the phosphorylation of mitogen-activated protein kinase (MAP kinase) in mouse CA1 hippocampal neurons, an effect that was blocked by scopolamine, confirming central M1 muscarinic receptor agonist activity in vivo. Lastly, an analysis of clozapine and NDMC serum levels in schizophrenic patients indicated that high NDMC/clozapine ratios better predicted improvement in cognitive functioning and quality of life than the levels of either compound alone. CONCLUSIONS: The muscarinic receptor agonist activities of NDMC are unique among antipsychotics, and provide a possible molecular basis for the superior clinical effects of clozapine pharmacotherapy.

beta-amyloid peptides enhance alpha-synuclein accumulation and neuronal deficits in a transgenic mouse model linking Alzheimer's disease and Parkinson's disease.

Alzheimer's disease and Parkinson's disease are associated with the cerebral accumulation of beta-amyloid and alpha-synuclein, respectively. Some patients have clinical and pathological features of both diseases, raising the possibility of overlapping pathogenetic pathways. We generated transgenic (tg) mice with neuronal expression of human beta-amyloid peptides, alpha-synuclein, or both. The functional and morphological alterations in doubly tg mice resembled the Lewy-body variant of Alzheimer's disease. These mice had severe deficits in learning and memory, developed motor deficits before alpha-synuclein singly tg mice, and showed prominent age-dependent degeneration of cholinergic neurons and presynaptic terminals. They also had more alpha-synuclein-immunoreactive neuronal inclusions than alpha-synuclein singly tg mice. Ultrastructurally, some of these inclusions were fibrillar in doubly tg mice, whereas all inclusions were amorphous in alpha-synuclein singly tg mice. beta-Amyloid peptides promoted aggregation of alpha-synuclein in a cell-free system and intraneuronal accumulation of alpha-synuclein in cell culture. beta-Amyloid peptides may contribute to the development of Lewy-body diseases by promoting the aggregation of alpha-synuclein and exacerbating alpha-synuclein-dependent neuronal pathologies. Therefore, treatments that block the production or accumulation of beta-amyloid peptides could benefit a broader spectrum of disorders than previously anticipated.

Role of apolipoprotein E receptors in regulating the differential in vivo neurotrophic effects of apolipoprotein E.

Apolipoprotein E (apoE) is known to bind to at least five receptors, including the low-density lipoprotein (LDL) receptor-related protein (LRP), very low density LDL receptor (VLDL-R), LDL-R, apoE receptor 2 (apoER2), and megalin/gp330. In this context, the main objective of the present study was to better understand the contributions of LRP and LDL-R to the in vivo neurotrophic effects of apoE. For this purpose, apoE-deficient and receptor-associated protein (RAP)-deficient mice were infused with recombinant apoE3, RAP, or saline. Infusion of apoE3 into apoE-deficient mice resulted in amelioration of degenerative alterations of pyramidal neurons, but had no effect on somatostatin-producing interneurons. In contrast, infusion of apoE3 into RAP-deficient mice resulted in amelioration of degenerative alterations of somatostatin-producing interneurons. LRP and LDL-R levels were significantly reduced in RAP-deficient mice, but significantly increased in the apoE-deficient mice. In contrast, levels of apoE were reduced in the RAP-deficient mice compared to wildtype controls, suggesting that neurotrophic effects of apoE3 in the RAP-deficient mice were related to a combined deficit in endogenous apoE and selected apoE receptors. Furthermore, in apoE-deficient mice, infusion of apoE3 had a neurotrophic effect on somatostatin-producing interneurons only when combined with RAP, suggesting that increased expression of apoE receptors in apoE-deficient mice prevented apoE from rescuing somatostatin-producing neurons. This study supports the contention that some of the in vivo neurotrophic effects of apoE are mediated by LRP and LDL-R and that a critical balance between levels of apoE and its receptors is necessary for the differential neurotrophic effects to appear.

Vitamin E supplementation prevents spatial learning deficits and dendritic alterations in aged apolipoprotein E-deficient mice.

Recent studies have suggested that altered function of apolipoprotein E might lead to Alzheimer's disease via oxidative stress. In this context, the objective of this study was to determine if antioxidative treatment with vitamin E was neuroprotective in apolipoprotein E-deficient mice. For this purpose, 1-month-old control and apolipoprotein E-deficient mice received dietary vitamin E for 12 months. We showed that, compared to apolipoprotein E-deficient mice who received a regular diet, mice treated with vitamin E displayed a significantly improved behavioural performance in the Morris water maze. This improved performance was associated with preservation of the dendritic structure in vitamin E-treated apolipoprotein E-deficient mice. In addition, whilst untreated apolipoprotein E-deficient mice displayed increased levels of lipid peroxidation and glutathione, vitamin E-treated mice showed near normal levels of both lipid peroxidation and glutathione. These results support the contention that vitamin E prevents the age-related neurodegenerative alterations in apolipoprotein E-deficient mice.

Neurotrophic effects of Cerebrolysin in animal models of excitotoxicity.

Excitotoxicity might play an important role in neurodegenerative disorders such as Alzheimer's disease. In the mouse brain, kainic acid (KA) lesioning results in neurodegeneration patterns similar to those found in human disease. For this study, two sets of experiments were performed in order to determine if Cerebrolysin ameliorates the alterations associated with KA administration. In the first set of experiments, mice received intraperitoneal KA injections followed by Cerebrolysin administration, while in the second, mice were pretreated with Cerebrolysin for 4 weeks and then challenged with KA. Behavioral testing in the water maze and assessment of neuronal structure by laser scanning confocal microscopy showed a significant protection against KA lesions in mice pretreated with Cerebrolysin. In contrast, mice that received Cerebrolysin after KA injections did not show significant improvement. This study supports the contention that Cerebrolysin might have a neuroprotective effect in vivo against excitotoxicity.

Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders.

To elucidate the role of the synaptic protein alpha-synuclein in neurodegenerative disorders, transgenic mice expressing wild-type human alpha-synuclein were generated. Neuronal expression of human alpha-synuclein resulted in progressive accumulation of alpha-synuclein-and ubiquitin-immunoreactive inclusions in neurons in the neocortex, hippocampus, and substantia nigra. Ultrastructural analysis revealed both electron-dense intranuclear deposits and cytoplasmic inclusions. These alterations were associated with loss of dopaminergic terminals in the basal ganglia and with motor impairments. These results suggest that accumulation of wild-type alpha-synuclein may play a causal role in Parkinson's disease and related conditions.

A role for apoE in regulating the levels of alpha-1-antichymotrypsin in the aging mouse brain and in Alzheimer's disease.

This study was designed to explore the possible functional relationships between apolipoprotein E (apoE) and the protease inhibitor alpha-1-antichymotrypsin in the aging mouse brain and in Alzheimer's disease. For this purpose, levels of EB22/5 (the mouse homologue to human alpha-1-antichymotrypsin) mRNA expression was studied in apoE-deficient mice. These mice showed an age-dependent increase of EB22/5 mRNA expression in the brain. Furthermore, overexpression of allele 3 of human APOE gene in transgenic mice (in an apoE-deficient background) resulted in normalization of levels of EB22/5 mRNA expression compatible with levels found in control mice. In contrast, overexpression of human APOE4 allele or down-regulation of the apoE receptor low density lipoprotein receptor-related protein by deletion of the receptor-associated protein was associated with elevated levels of EB22/5 similar to apoE-deficient mice. Consistent with the findings in murine models, human alpha-1-antichymotrypsin protein was increased in brain homogenates from patients with Alzheimer's disease, and levels of this serpin were the highest in patients with the APOE4 allele. In summary, the present study showed evidence supporting a role for apoE in regulating alpha-1-antichymotrypsin expression. This is relevant to Alzheimer's disease because these two molecules appear to be closely associated with the pathogenesis of this disorder.

Synaptotagmin and synaptic transmission alterations in apolipoprotein E-deficient mice.

1. Aged apoE-deficient mice and age-matched controls were tested for cognitive alterations in the Morris water maze. 2. Water maze results were correlated with in vivo electrophysiology and expression of the synaptic protein synaptotagmin (p65). 3. Compared to age-matched controls, apolipoprotein E-deficient mice displayed significant performance impairment accompanied by in vivo electrophysiological alterations in the dentate gyrus. 4. Apolipoprotein E-deficient mice also showed a significant increase in the synaptic protein, synaptotagmin, a synaptic calcium sensor involved in neurotransmitter release. 5. Cognitive impairments in these animals may be associated with decreased synaptic excitability in hippocampal neurons and the regulatory role of apolipoprotein E in synaptic function might be mediated by modulation of the expression of calcium sensor proteins.

A novel role for receptor-associated protein in somatostatin modulation: implications for Alzheimer's disease.

Receptor-associated protein appears to play an important role in low-density lipoprotein receptor-related protein trafficking. Since ligands for the low-density lipoprotein receptor-related protein have been implicated in Alzheimer's disease and normal functioning of this protein is indispensable for central nervous system development, deficient receptor-associated protein expression may result in central nervous system alterations. In this study, receptor-associated protein knockout mice were behaviorally tested and nervous system integrity was assessed via in situ hybridization and immunocytochemical/laser confocal microscopy methods. Receptor-associated protein knockout mice were found to be cognitively impaired in the Morris water maze compared to controls. In wild-type mice, the receptor-associated protein was found to be highly co-expressed with somatostatin in hippocampal and neocortical inhibitory neurons. Receptor-associated protein knockout mice, however, showed a significant decrease in number of somatostatin-expressing neurons of the CA1 region and somatostatin expression within these neurons. The decreased number of somatostatin neurons significantly correlated with cognitive impairment observed in the receptor-associated protein knockout mice. These results suggest a novel role for receptor-associated protein in modulating the functioning of somatostatin-producing neurons. Furthermore, this has implications for Alzheimer's disease pathogenesis, in which altered regulation of both somatostatin and the known low-density lipoprotein receptor-related protein ligands are a consistent finding.

Differential neurotrophic effects of apolipoprotein E in aged transgenic mice.

The present study seeked to determine whether the neurodegenerative and cognitive alterations in aged apolipoprotein E-deficient mice are differentially reversed by transgenic overexpression of human apolipoprotein-E3 vs. apolipoprotein-E4 in the background of deficient endogenous apolipoprotein E. These studies showed dendritic alterations in pyramidal neurons of apolipoprotein-E4 transgenic mice, similar to the ones observed in apolipoprotein E-deficient mice. However, these mice had a preserved density of synaptophysin-immunoreactive presynaptic terminals. In contrast, mice overexpressing apolipoprotein-E3 showed no synapto-dendritic alterations. Analysis of behavioral performance in the Morris water maze showed that while apolipoprotein E-deficient mice performed poorly, overexpression of apolipoprotein-E3 and, to a lower extent apolipoprotein-E4, resulted in an improved performance. This study supports the contention that, compared with apolipoprotein-E4, apolipoprotein-E3 might have a greater neurotrophic in vivo effect in aged mice.

Cerebrolysin ameliorates performance deficits, and neuronal damage in apolipoprotein E-deficient mice.

Recent studies suggest that Cerebrolysin improves behavioral performance by affecting synaptic transmission in the hippocampus. The main objective of this study was to determine if Cerebrolysin administration ameliorates the neurodegenerative and performance deficits in aged apolipoprotein E (apoE)-deficient mice. ApoE-deficient mice treated with Cerebrolysin showed a significant improved performance in the Morris water maze, compared to saline-treated apoE-deficient mice. Although the improved performance in the Cerebrolysin-treated apoE-deficient mice was associated with restoration of the neuronal structure, the poor learning ability of saline-treated apoE-deficient mice was related to the a disrupted synaptodendritic structure. This study supports the contention that Cerebrolysin might have a neurotrophic effect in vivo.

Altered long-term potentiation in the hippocampus of apolipoprotein E-deficient mice.

Recent studies suggest that apolipoprotein E (apoE) plays a neurotrophic role in the central nervous system and that an aberrant function of this molecule might result in neurodegeneration. Supporting this notion, apoE-deficient mice show neurodegenerative and cognitive alterations. To characterize physiological changes associated with synaptic damage and cognitive impairment in apoE-deficient mice, we investigated synaptic plasticity in the hippocampus of urethane anesthetized mice. Electrical stimulation was delivered to the perforant pathway and the resulting evoked field excitatory postsynaptic potential (EPSP) and population spike were recorded in the hilus. Long-term potentiation, as measured in the population spike, was reduced by 50% in apoE-deficient mice when compared to wild-type controls. In contrast, there were no significant differences in the evoked field EPSP between wild-type and apoE-deficient mice following high-frequency stimulation. These results support the notion that cognitive impairment and synaptic loss in the hippocampus of apoE-deficient mice might be associated with impaired long-term potentiation.

Expression pattern of synucleins (non-Abeta component of Alzheimer's disease amyloid precursor protein/alpha-synuclein) during murine brain development.

The non-Abeta component of Alzheimer's disease amyloid precursor protein (NACP) is predominantly a neuron-specific presynaptic protein that may play a central role in neurodegeneration because NACP fragments are found in Alzheimer's disease amyloid and a mutation in the NACP gene is associated with familial Parkinson's disease. In addition, NACP may play an important role during synaptogenesis and CNS development. To understand better the patterns of NACP expression during development, we analyzed the levels of this protein as well as the levels of another synaptic protein (synaptophysin) by ribonuclease protection assay, western blotting, and immunocytochemistry in fetal, juvenile, and adult mouse brain. From embryonic day 12 to 15, there was a slight increase, which was then followed by a more dramatic increase at later time points. Immunocytochemical staining for NACP increases throughout these stages as well. Although NACP appeared early in CNS development, synaptophysin levels started to rise at a later stage. These findings support the contention that NACP might be important for CNS development. Furthermore, the cytosolic component of NACP precedes the particulate component in development, indicating that a redistribution of the protein to the membrane fraction may be important for events later in neuronal development and in synaptogenesis.

Neurodegeneration and cognitive impairment in apoE-deficient mice is ameliorated by infusion of recombinant apoE.

Recent studies suggest that apolipoprotein E (apoE) might play a neurotrophic function in the central nervous system and that altered functioning of this molecule could result in neurodegeneration. The main objective of this study was to determine if neurodegenerative and cognitive alterations in apoE-deficient mice are reversible by infusion of recombinant apoE into the lateral ventricles. ApoE-deficient mice treated with either apoE3 or apoE4 showed a significant improvement in their learning capacity in the Morris water maze compared to saline-infused apoE-deficient mice. While this improved performance was associated with restoration of neuronal structure, the poor learning ability of apoE-deficient mice treated with saline correlated with the disrupted synapto-dendritic structure. This study supports the contention that apoE might play a neurotrophic effect in vivo and suggests that apoE might have a potential therapeutic role.

Alterations in apolipoprotein E expression during aging and neurodegeneration.

Apolipoprotein E (apoE) is a 34 kDa protein that plays an important role in cholesterol transport, uptake and redistribution. Within the nervous system, apoE might be involved in maintaining synaptic integrity after injury and during aging. ApoE might help maintain the integrity of the synaptodendritic complex by several different mechanisms. Among them, recent studies have suggested that apoE: (1) stabilizes the neuronal cytoskeleton; (2) plays an important role in transporting esterified cholesterol to neurons undergoing reinnervation where it is taken up by the low density lipoprotein receptor-related protein pathway and used as a precursor for the synthesis of new synaptic terminals; (3) regulates interactions between neurons and the extracellular matrix (e.g. laminin); and (4) regulates levels of intracellular calcium. The main objective of the manuscript is to review the current progress in understanding the functions of apoE in the nervous system and how malfunctioning of this molecule might result in neurodegenerative disorders such as Alzheimer's disease.

Neurodegeneration in the central nervous system of apoE-deficient mice.

Apolipoprotein E (apoE) is involved in the development and regeneration of the central nervous system (CNS). ApoE may also be necessary to maintain the integrity of the synapto-dendritic complexity. We analyzed the synaptic alterations in the CNS of apoE-deficient (knockout) mice during the aging process. In apoE-deficient homozygous mice, there was an age-dependent 15 to 40% loss of synaptophysin-immunoreactive nerve terminals and microtubule-associated protein 2-immunoreactive dendrites in the neocortex and hippocampus, when compared to controls. Dendritic alterations were observed as early as 4 months of age. Ultrastructural analysis revealed extensive dendritic vacuolization and disruption of the endomembrane system and cytoskeleton in apoE-deficient homozygous mice. Further immunocytochemical studies of the neuronal cytoskeleton showed that in apoE-deficient mice there was a decrease in the immunoreactivity of alpha and beta tubulin (but not kinesin) in the cell bodies and processes. These results support the contention that apoE might play an important role in maintaining the stability of the synapto-dendritic apparatus and that altered or deficient functioning of this molecule could underlie the synaptic and cytoskeletal alterations in Alzheimer's disease.

Increase of synaptic density and memory retention by a peptide representing the trophic domain of the amyloid beta/A4 protein precursor.

The secreted form (sAPP) of the Alzheimer amyloid beta/A4 protein precursor (APP) has been shown to be involved in the in vitro regulation of fibroblast growth and neurite extension from neuronal cells. The active site of sAPP responsible for these functions is within a small domain just C-terminal to the Kunitz-type protease inhibitor (KPI) insertion site. We report here that a 17-mer peptide, containing this active domain of sAPP, can induce cellular and behavioral changes when infused into rat brains. After 2 weeks of APP 17-mer peptide infusion, the animals were tested for reversal learning and memory retention and were sacrificed for morphological examination of brains. We found that administration of the APP 17-mer peptide resulted in an 18% increase in the number of presynaptic terminals in the frontoparietal cortex. At the behavioral level, 17-mer-infused animals with nonimpaired learning capability showed an increased memory retention that seemed to interfere with reversal learning performance. This APP 17-mer effect on memory retention was not observed in animals with impaired initial learning capacity. These results suggest that APP is involved in memory retention through its effect on synaptic structure.